The regulatory process known as protein ubiquitination modifies cellular proteins with far-reaching impacts on human health and disease. It is involved in every known biological process and is implicated in a growing range of diseases that includes cancers, neurodegenerative diseases, muscle wasting, etc. We are studying the expanding roles of protein ubiquitination to understand underlying principles of the process that can guide future efforts to manipulate or target the process. Over its thirteen-year course, the scope of the project has expanded beyond the single ubiquitin E3 ligase, the breast cancer susceptibility protein, BRCA1/BARD1, and its E2 conjugating enzymes. To reflect our expanding field of investigation and the expanding functionalities of E2s and E3s we have uncovered, the project is renamed Expanding Roles of E2 and E3 enzymes in Ubiquitin Transfer. In Aim 1, we will use new mechanistic insights and new technology to generate RING E3s with enhanced activities for use in functional studies. In Aim 2, we will investigate the RING-Between-RING E3s, newly identified as a novel RING-HECT hybrid class of E3s. In Aim 3, we seek to define novel mechanisms used by E2s.